The fungal type II myosin in Penicillium marneffei, MyoB, is essential for chitin deposition at nascent septation sites but not actin localization

Cytokinesis is essential for proliferative growth but also plays equally important roles during morphogenesis and development. The human pathogen Penicillium marneffei is capable of dimorphic switching in response to temperature, growing in a multicellular filamentous hyphal form at 25°C and in a unicellular yeast form at 37°C. P. marneffei also undergoes asexual development at 25°C to produce multicellular differentiated conidiophores. Thus, P. marneffei exhibits cell division with and without cytokinesis and division by budding and fission, depending on the cell type. The type II myosin gene, myoB, from P. marneffei plays important roles in the morphogenesis of these cell types. Deletion of myoB leads to chitin deposition defects at sites of cell division without perturbing actin localization. In addition to aberrant hyphal cells, distinct conidiophore cell types are lacking due to malformed septa and nuclear division defects. At 37°C, deletion of myoB prevents uninucleate yeast cell formation, instead producing long filaments resembling hyphae at 25°C. The ΔmyoB cells also often lyse due to defects in cell wall biogenesis. Thus, MyoB is essential for correct morphogenesis of all cell types regardless of division mode (budding or fission) and defines differences between the different types of growth.     

A basic helix-loop-helix protein with similarity to the fungal morphological regulators, Phd1p, Efg1p and StuA, controls conidiation but not dimorphic growth in Penicillium marneffei

Members of the APSES protein group are basic helix-loop-helix (bHLH) proteins that regulate processes such as mating, asexual sporulation and dimorphic growth in fungi. Penicillium marneffei is a human pathogen and is the only member of its genus to display a dimorphic growth transition. At 25 degrees C, P. marneffei grows with a filamentous morphology and produces asexual spores from multicellular con-idiophores. At 37 degrees C, the filamentous morphology is replaced by yeast cells that reproduce by fission. We have cloned and characterized an APSES protein-encoding gene from P. marneffei that has a high degree of similarity to Aspergillus nidulans stuA. Deletion of stuA in P. marneffei showed that it is required for metula and phialide formation during conidiation but is not required for dimorphic growth. This suggests that APSES proteins may control processes that require budding (formation of the metulae and phialides, pseudohyphal growth in Saccharomyces cerevisiae and dimorphic growth in Candida albicans) but not those that require fission (dimorphic growth in P. marneffei). The A. nidulans DeltastuA mutant has defects in both conidiation and mating. The P. marneffei stuA gene was capable of complementing the conidiation defect but could only inefficiently complement the sexual defects of the A. nidulans mutant. This suggests that the P. marneffei gene, which comes from an asexual species, has diverged significantly from the A. nidulans gene with respect to sexual but not asexual development.     

An STE12 homolog from the asexual, dimorphic fungus Penicillium marneffei complements the defect in sexual development of an Aspergillus nidulans steA mutant

Penicillium marneffei is an opportunistic fungal pathogen of humans and the only dimorphic species identified in its genus. At 25 degrees P. marneffei exhibits true filamentous growth, while at 37 degrees P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. Members of the STE12 family of regulators are involved in controlling mating and yeast-hyphal transitions in a number of fungi. We have cloned a homolog of the S. cerevisiae STE12 gene from P. marneffei, stlA, which is highly conserved. The stlA gene, along with the A. nidulans steA and Cryptococcus neoformans STE12alpha genes, form a distinct subclass of STE12 homologs that have a C2H2 zinc-finger motif in addition to the homeobox domain that defines STE12 genes. To examine the function of stlA in P. marneffei, we isolated a number of mutants in the P. marneffei-type strain and, in combination with selectable markers, developed a highly efficient DNA-mediated transformation procedure and gene deletion strategy. Deletion of the stlA gene had no detectable effect on vegetative growth, asexual development, or dimorphic switching in P. marneffei. Despite the lack of a detectable function, the P. marneffei stlA gene complemented the sexual defect of an A. nidulans steA mutant. In addition, substitution rate estimates indicate that there is a significant bias against nonsynonymous substitutions. These data suggest that P. marneffei may have a previously unidentified cryptic sexual cycle.     

Insights into the pathogenicity of Penicillium marneffei.

Penicillium marneffei is a significant pathogen of AIDS patients in Southeast Asia. This fungus is unique in that it is the only dimorphic member of the genus. Pathogenesis of P. marneffei requires the saprobic mold form to undergo a morphological change upon tissue invasion. The in vivo form of this fungus reproduces as a fission yeast that capably evades the host immune system. The processes that control these morphological changes, better termed as phase transition, can be replicated in vitro by incubation of the mold form at 37 degrees C. The unidentified molecular mechanisms regulating phase transition in this fungus are now being uncovered using modern methodologies and novel strategies. A better comprehension of these underlying regulatory pathways will provide insight into eukaryotic cellular development as well as the potential factors responsible for infections caused by P. marneffei and other fungi. Such knowledge may lead to better chemotherapeutic interventions of fungal diseases.     

Photosynthetic and respiratory acclimation and growth response of Antarctic vascular plants to contrasting temperature regimes

Air temperatures have risen over the past 50 yr along the Antarctic Peninsula, and it is unclear what impact this is having on Antarctic plants. We examined the growth response of the Antarctic vascular plants Colobanthus quitensis (Caryophyllaceae) and Deschampsia antarctica (Poaceae) to temperature and also assessed their ability for thermal acclimation, in terms of whole-canopy net photosynthesis (P(n)) and dark respiration (R(d)), by growing plants for 90 d under three contrasting temperature regimes: 7°C day/7°C night, 12°C day/7°C night, and 20°C day/7°C night (18 h/6 h). These daytime temperatures represent suboptimal (7°C), near-optimal (12°C), and supraoptimal (20°C) temperatures for P(n) based on field measurements at the collection site near Palmer Station along the west coast of the Antarctic Peninsula. Plants of both species grown at a daytime temperature of 20°C had greater RGR (relative growth rate) and produced 2.2-3.3 times as much total biomass as plants grown at daytime temperatures of 12° or 7°C. Plants grown at 20°C also produced 2.0-4.1 times as many leaves, 3.4-5.5 times as much total leaf area, and had 1.5-1.6 times the LAR (leaf area ratio; leaf area:total biomass) and 1.1-1.4 times the LMR (leaf mass ratio; leaf mass:total biomass) of plants grown at 12° or 7°C. Greater RGR and biomass production at 20°C appeared primarily due to greater biomass allocation to leaf production in these plants. Rates of P(n) (leaf-area basis), when measured at their respective daytime growth temperatures, were highest in plants grown at 12°C, and rates of plants grown at 20°C were only 58 (C. quitensis) or 64% (D. antarctica) of the rates in plants grown at 12°C. Thus, lower P(n) per leaf area in plants grown at 20°C was more than offset by much greater leaf-area production. Rates of whole-canopy P(n) (per plant), when measured at their respective daytime growth temperatures, were highest in plants grown at 20°C, and appeared well correlated with differences in RGR and total biomass among treatments. Colobanthus quitensis exhibited only a slight ability for relative acclimation of P(n) (leaf-area basis) as the optimal temperature for P(n) increased from 8.4° to 10.3° to 11.5°C as daytime growth temperatures increased from 7° to 12° to 20°C. There was no evidence for relative acclimation of P(n) in D. antarctica, as plants grown at all three temperature regimes had a similar optimal temperature (10°C) for P(n). There was no evidence for absolute acclimation of P(n) in either species, as rates of P(n) in plants grown at a daytime temperature of 12°C were higher than those of plants grown at daytime temperatures of 7° or 20°C, when measured at their respective growth temperatures. The poor ability for photosynthetic acclimation in these species may be associated with the relatively stable maritime temperature regime during the growing season along the Peninsula. In contrast to P(n), both species exhibited full acclimation of R(d), and rates of R(d) on a leaf-area basis were similar among treatments when measured at their respective daytime growth temperature. Our results suggest that in the absence of interspecific competition, continued warming along the Peninsula will lead to improved vegetative growth of these species due to (1) greater biomass allocation to leaf-area production (as opposed to improved rates of P(n) per leaf area) and (2) their ability to acclimate R(d), such that respiratory losses per leaf area do not increase under higher temperature regimes.     

Studies on the role of the areA gene in the regulation of nitrogen catabolism in Aspergillus nidulans.

Mutants of Apergillus nidulans with lesions in a gene, areA (formerly called amdT), have been isolated by a variety of different selection methods. The areA mutants show a range of pleiotropic growth responses to a number of compounds as sole nitrogen sources, but are normal in utilization of carbon sources. The levels of two amidase enzymes as well as urease have been investigated in the mutants and have been shown to be affected by this gene. Most of the areA mutants have much lower amidase-specific activities when grown in ammonium-containing medium, compared with mycelium incubated in medium lacking a nitrogen source. Some of the areA mutants do not show derepression of urease upon relief of ammonium repression. The dominance relationships of areA alleles have been investigated in heterozygous diploids, and these studies lend support to the proposal that areA codes for a positively acting regulatory product. One of the new areA alleles is partially dominant to areA+ and areA102. This may be a result of negative complementation or indicate that areA has an additional negative regulatory function. Investigation of various amdR; areA double mutants has led to the conclusion that amdR and areA participate in independent regulatory circuits in the control of acetamide utilization. Studies on an amdRc; areA double mutant indicate that areA is involved in derepression of acetamidase upon relief of ammonium repression.     

Usefulness of a microimmunodiffusion test for the detection of Penicillium marneffei antigenemia,antibodies, and exoantigens

Eight sera from culturally-proven cases of penicilliosis marneffei and their corresponding isolates were examined for circulating antibody(ies) and antigen, and exoantigens, respectively, using a microimmunodiffusion (MID) test. Two of the 8 sera produced strong precipitins (1-2) when reacted against control Penicillium marneffei antigen (5-week-old shaken cultures at 25 C) in the presence of control rabbit anti-P. marneffei serum. Five of the 8 sera produced a strong precipitin line when reacted against control hyperimmune serum to P. marneffei. These five sera, and one additional serum, which tested negative for antibody to P. marneffei, demonstrated the presence of antigen by reacting only against the anti-P. marneffei serum. Serological evaluations of the sera revealed that the MID test is capable of detecting antibody and antigen in AIDS patients having penicilliosis marneffei infections. Exoantigen analysis of the 8 P. marneffei isolates, which were previously identified using this conventional and time-consuming macro- and micro-morphological characteristics, showed the presence of 1 to 4 specific exoantigens in MID. With the exoantigen analysis, the identity of all of the isolates was confirmed as P. marneffei. Our studies indicated that the serological tests are useful for detecting circulating antibody and/or antigen in patients' sera, and that the exoantigen test is reliable for confirming the identity of P. marneffei cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

Penicillium marneffei: an insurgent species among the penicillia

Most species of Penicillium are considered relatively benign with respect to causing human disease. However, one species, P. marneffei, has emerged as a significant pathogen particularly among individuals who live in Southeast Asia and are concurrently infected with the human immunodeficiency virus. While environmental and epidemiological studies have yet to resolve the reason for the heightened virulence of P. marneffi, one characteristic does distinguish this fungus from other Penicillium species. Whereas the latter grow as monomorphic moulds bearing typical asexual propagules (conidia), P. marneffei is thermally dimorphic. At room temperature, P. marneffei exhibits the morphology characteristic of the genus. In contrast to other Penicillia, though, P. marneffei grows as a yeast-like entity (arthroconidium) when found in diseased tissue or cultivated at 37 degrees C. Studies in our laboratory have focused on the differential gene expression between the mould and arthroconidial phases. Many of the genes whose expression differs during mould-to-arthrocondium transition are related to energy metabolism. A better understanding of gene expression during morphogenesis in P. marneffei may help detect unique target sites or cellular processes that can be exploited in the development of antifungal agents or immunomodulation therapies.     

Sequential temperature control of multi-phasic dormancy release and germination of Paeonia corsica seeds

Aims The physiological responses during dormancy removal and multi-phasic germination were investigated in seeds of Paeonia corsica (Paeoniaceae).Methods Seeds of P. corsica were incubated in the light at a range of temperatures (10–25 and 25/10°C), without any pre-treatment, after W (3 months at 25°C), C (3 months at 5°C) and W + C (3 months at 25°C followed by 3 months at 5°C) stratification, and a GA 3 treatment (250 mg·l^-1 in the germination substrate). Embryo growth, time from testa to endosperm rupture and radicle emergence were assessed as separate phases. Epicotyl–plumule emergence was evaluated incubating the germinated seeds at 15°C for 2 weeks, at 5 and 25°C for 2 months on agar water before transplanting to the soil substrate at 10, 15 and 20°C and at 15°C for 2 months on the surface agar water with GA 3 .Important findings Embryo growth, testa rupture, endosperm rupture (radicle emergence) and growth of the epicotyl were identified as four sequential steps in seeds of P. corsica. Gibberellic acid alone and warm stratification followed by 15°C promoted embryo growth and subsequent seed germination. Cold stratification induced secondary dormancy, even when applied after warm stratification. After radicle emergence, epicotyl–plumule emergence was delayed for ca. 3 months. Mean time of epicotyl–plumule emergence was positively affected by cold stratification (2 months at 5°C) and GA 3. P. corsica seeds exhibited differential temperature sensitivity for the four sequential steps in the removal of dormancy and germination processes that resulted in the precise and optimal timing of seedling emergence.     

温度对青蒿毛状根生长和青蒿素生物合成的影响

本实验研究了不同温度(15℃～35℃)对青蒿毛状根生长和青蒿素生物合成的影响,发现25℃有利于毛状根生长,30℃促进了青蒿素生物合成。通过温度改变的二步培养技术(培养前20d温度控制在25℃,后10d温度提高到30℃),青蒿素的产量得到明显提高,高于在恒温培养时(25℃或30℃)的结果。     

Genetic analysis of growth, morphology and pathogenicity in the F(1) progeny of an interspecific cross between Fusarium circinatum and Fusarium subglutinans

Fusarium circinatum and Fusarium subglutinans are two distinct species in the Gibberella fujikuroi species complex. A genetic linkage map produced from an interspecific cross between these species was used to identify quantitative trait loci (QTLs) associated with variation in mycelial growth and morphology of colony margins (CMs) in the 94 F(1) progeny. Mycelial growth was assessed by measuring culture size at 25°C and 30°C, while CM morphology was characterized in the parents and assessed in their F(1) progeny. In order to test the pathogenicity of the progeny, Pinus patula seedlings were inoculated and lesion lengths were measured after 3weeks. Seven putative QTLs were associated with mycelial growth, three for growth at 25°C and four at 30°C. One highly significant QTL (P<0.001) was present at both growth temperatures. For CM morphology, a QTL was identified at the same position (P<0.001) as the QTL responsible for growth at the two temperatures. The putative QTLs accounted for 45 and 41% of the total mycelial growth variation at 25°C and 30°C, respectively, and for 21% of the variation in CM morphology. Only one of the 94 F(1) progeny was pathogenic on P. patula seedlings. This observation could be explained by the genetic constitution of this F(1) isolate, namely that ～96% of its genome originated from the F. circinatum parent. This F(1) individual also grew significantly faster at 25°C than the F. circinatum parent (P<0.05), as well as more rapidly than the average growth for the remaining 93 F(1) progeny (P<0.05). However, no association was found between mycelial growth and pathogenicity at 25°C. The highly significant QTL associated with growth at two temperatures, suggests that this is a principal genomic region involved in mycelial growth at both temperatures, and that the same region is also responsible for CM morphology.     

Exploring the<Emphasis Type="Italic"> Penicillium marneffei</Emphasis> genome

Penicillium marneffei is a dimorphic fungus that intracellularly infects the reticuloendothelial system of humans and bamboo rats. Endemic in Southeast Asia, it infects 10% of AIDS patients in this region. The absence of a sexual stage and the highly infectious nature of the mould-phase conidia have impaired studies on thermal dimorphic switching and host-microbe interactions. Genomic analysis, therefore, could provide crucial information. Pulsed-field gel electrophoresis of genomic DNA of P. marneffei revealed three or more chromosomes (5.0, 4.0, and 2.2 Mb). Telomeric fingerprinting revealed 6-12 bands, suggesting that there were chromosomes of similar sizes. The genome size of P. marneffei was hence about 17.8-26.2 Mb. G+C content of the genome is 48.8 mol%. Random exploration of the genome of P. marneffei yielded 2303 random sequence tags (RSTs), corresponding to 9% of the genome, with 11.7, 6.3, and 17.4% of the RSTs having sequence similarity to yeast-specific sequences, non-yeast fungus sequences, and both (common sequences), respectively. Analysis of the RSTs revealed genes for information transfer (ribosomal protein genes, tRNA synthetase subunits, translation initiation, and elongation factors), metabolism, and compartmentalization, including several multi-drug-resistance protein genes and homologues of fluconazole-resistance gene. Furthermore, the presence of genes encoding pheromone homologues and ankyrin repeat-containing proteins of other fungi and algae strongly suggests the presence of a sexual stage that presumably exists in the environment.     

Mycorrhizal benefit in two low arctic herbs increases with increasing temperature

Climate change may influence the relationship between arctic plants and their symbiotic mycorrhizal fungi. The benefit of the symbiosis for the host plant affects vegetation succession and may be a key parameter in predicting vegetation responses to warming. We investigated the mycorrhizal benefit in the low arctic perennial herbs Potentilla crantzii and Ranunculus acris in symbiosis with the arbuscular mycorrhizal fungus Glomus claroideum. Temperature response in the mycorrhiza-mediated acquisition of nitrogen (N) and phosphorus (P), growth, and photosynthetic nutrient-use efficiency were determined. Near the average natural soil temperature (12°C), mycorrhiza did not improve plant nutrient capture but significantly enhanced plant P capture at 17°C. Photosynthetic nitrogen-use efficiency was higher at 17°C than at 12°C and was further increased by mycorrhiza at 17°C. Photosynthetic phosphorus-use efficiency was not affected by temperature or mycorrhiza. Increasing the growing temperature by 5°C increased the relative shoot growth rate by 15%. Mycorrhizal symbiosis did not enhance plant growth rate, but the plants gained between 20% and 90% more mycorrhiza-mediated P when grown at higher temperature. The results suggest that these low arctic species have good potential to respond positively to increasing temperatures.     

Effect of temperature level on thermal acclimation in Large White growing pigs

The effect of temperature level (24°C, 28°C, 32°C or 36°C) on performance and thermoregulatory response in growing pigs during acclimation to high ambient temperature was studied on a total of 96 Large White barrows. Pigs were exposed to 24°C for 10 days (days -10 to -1, P0) and thereafter to a constant temperature of 24°C, 28°C, 32°C or 36°C for 20 days. Pigs were housed in individual metal slatted pens, allowing a separate collection of faeces and urine and given ad libitum access to feed. Rectal (RT) and cutaneous (CT) temperatures and respiration rate (RR) were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. From day 1 to 20, the effect of temperature on average daily feed intake (ADFI) and BW gain (average daily gain, ADG) was curvilinear. The decrease of ADFI averaged 90 g/day per °C between 24°C and 32°C and 128 g/day per °C between 32°C and 36°C. The corresponding values for ADG were 50 and 72 g/day per °C, respectively. The 20 days exposure to the experimental temperature was divided in two sub-periods (P1 and P2, from day 1 to 10 and from day 11 to 20, respectively). ADFI was not affected by duration of high-temperature exposure (i.e. P2 v. P1). The ADG was not influenced by the duration of exposure at 24°C and 28°C groups. However, ADG was higher at P2 than at P1 and this effect was temperature dependent (+130 and +458 g/day at 32°C and 36°C, respectively). In P2 at 36°C, dry matter digestibility significantly increased (+2.1%, P < 0.01); however, there was no effect of either duration or temperature on the digestibility of dry matter at group 24°C and 32°C. RT, CT and RR were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. Between 28°C and 36°C, RT and CT were lower during P2 than during P1 (-0.20°C and -0.23°C; P < 0.05), whereas RR response was not affected by the duration of exposure whatever the temperature level. In conclusion, this study suggests that the effect of elevated temperatures on performance and thermoregulatory responses is dependent on the magnitude and the duration of heat stress.